非整合型乙型肝炎病毒持续自主复制细胞模型的构建

The current widely-used cell lines with stable replication of Hepatitis B virus (HBV) are all characterized with HBV integration in cell genome, in which the HBV cannot be eliminated. Our research team had successfully constructed a replication-competent HBV vector with gene engineering procedures. Briefly, the over-lapping regions of C gene and P gene in HBV genome was separated and complemented to form two independent and complete open reading frames of HBV C gene and P gene. An additional short internal ribosome entry sites (IRES) was inserted before P gene to sustain its expression. When exogenous genes were subcloned into the newly-formed gap between C and P genes, they were expressed without obvious impairment of HBV replication. Based on our replication-competent HBV vector, we initiated a project composed of three parts. ① The replication-competent HBV vector will be inserted with blasticidin resistance gene BsdR or miniSOG fluorescence gene of 106 amino acids respectively, along with a SV40 replication origin of 64 base pairs. Then the recently-developed Minicircle DNA system will be utilized to generate circular double-stranded HBV. ② The hepatoma cell lines, HepG2T, will be constructed with stable expression of SV40 large T antigen. ③ The circular HBV DNA gained in the first part will be transfected into HepG2T cells to form the extrachromosomal HBV episomal genome (similar to HBV covalently closed circular DNA). After screening with Blasticidin or fluorecence observation, a novel cell model with stable and sustained HBV replication, without cellular chromosomal integration, will be constructed. This project is expected to generate a novel kind of HBV replication cell model, which is more similar with HBV natural replication circle than the current available HBV replication cell models, and will contribute to the study of HBV molecule biology and the development of anti-HBV drugs.

目前常用的HBV稳定复制细胞系均为基因整合型，不可能完全清除HBV。本实验室前期研究构建了一种复制型HBV载体：把HBV C、P基因重叠区分开表达，并插入小IRES增强P蛋白表达，在C、P基因之间插入外源基因后可顺利表达，同时保留了HBV复制能力。在此基础上，本研究分三部分：①应用这一复制型HBV载体分别表达抗生素筛选标志BsdR或106个氨基酸的miniSOG荧光基团，并插入一个64bp SV40复制起始序列，在此基础上，利用Minicircle DNA最新进展制备环状HBV DNA；②构建稳定表达SV40大T抗原的肝癌细胞系HepG2T；③环状HBV DNA转染HepG2T细胞系后形成染色体外游离HBV基因（类似于HBV cccDNA），获得非整合型HBV持续自主复制细胞系。本研究有望提供一个新型的，更接近HBV自然复制周期的细胞模型，将有助于HBV研究与药物筛选。

目前常用的HBV稳定复制细胞系均为基因整合型，不可能完全清除HBV，不能模拟HBV的自然复制周期，不利于筛选新型抗HBV药物。本实验室前期研究构建了一种复制型HBV载体：把HBV C、P基因重叠区分开表达，并插入两个串联的，短小的Rbm3 IRES，分别启动P基因和在C、P基因之间插入的外源基因的翻译过程，同时保留了重组HBV的复制能力。在此基础上，本研究分三部分展开了研究：i 在HCV复制子上插入两个串联的Rbm3 IRES，分别启动外源基因和HCV 结构基因的翻译过程，进而证实这样串联两个Rbm3 IRES足以表达外源基因，同时不影响载体的复制潜力；在此基础上，我们应用这一复制型HBV载体分别表达了抗生素筛选标志BsdR或miniSOG荧光基团，并插入一个64bp SV40复制起始序列，进而，利用minicircle DNA制备了小环状HBV DNA；ii构建了稳定表达SV40大T抗原的肝癌细胞系HepG2T；iii小环状HBV DNA转染HepG2T细胞系后形成染色体外游离HBV基因（类似于HBV cccDNA），进而获得了非整合型HBV持续自主复制细胞系。本研究有望提供一个新型的，更接近HBV自然复制周期的细胞模型，将有助于HBV研究与药物筛选，尤其适合用于探索能够作用于HBV cccDNA的新型药物。而HBV cccDNA正是彻底清除体内HBV的难点和关键所在。